The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Over the last year we made progress in defining the role of the inhibitory receptor, FcgammaRIIB, on regulating B cell receptor (BCR) signaling in human IgM-expressing naive B cells and in IgG-expressing memory B cells (MBCs). In addition we initiated studies to determine the function of the Fc like receptor 4 (FcRL4) present on a distinct subset of MBCs observed to be greatly expanded in HIV infected viremic individuals and in children and adults in malaria-endemic areas. Our progress using high resolution live cell imaging to delineate the very early antigen driven events in B cell activation has provided a new context in which the impact of coreceptors can be evaluated. Using high resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) and single molecule tracking we provided evidence for an ordered process that occurs within seconds to minutes of the BCR binding antigen. Antigen bound BCRs form immobile clusters that then grow in size by molecular trapping. The clusters perturb the local lipid environment causing lipid rafts to coalesce around the BCR clusters. As a consequence of the membrane perturbation the first kinase in the pathway, Lyn, that is tethered to the membrane by raft lipids is brought into close molecular proximity to the BCR clusters. Simultaneously, Lyn phosphorylates the Ig alpha beta cytoplasmic domain of the BCR and the Ig alpha beta chains undergo a conformational change from a closed to an open form. Syk is recruited to the phosphorylated BCR and the signaling cascades are triggered. Over the last year we initiated studies to define the function of FcRL4, a member of an ancient family of transmembrane proteins, the FcRLs, that share ancestors with the classical FcRs, like FcgammaRIIB, and are preferentially expressed in the B cell lineage. FcRL4 is expressed on a distinctive subset of MBCs located in mucosal lymphoid tissues in healthy individuals. In addition, FcRL4+ MBCs are greatly expanded in numbers in the blood of HIV-infected viremic individuals and in individuals chronically re-infected with malaria. We provided evidence that the expression of FcRL4 in human B cell lines inhibits antigen-induced BCR signaling at the level of Syk phosphorylation. Inhibition did not require coligation of the FcRL4 with the BCR but depended on the two immunoreceptor inhibitory motifs (ITIMs) in FcRL4s cytoplasmic domain. Remarkably, FcRL4 expression simultaneously enhanced signaling through the innate immune toll-like receptor 9 (TLR9). These findings suggest that FcRL4 may act as molecular switch in B cells to dampen adaptive immune signaling and enhance signaling in response to chronic antigenic stimulation. In collaboration with Dr. Susan Moir in NIAID we provided evidence that down regulation of FcRL4 in B cells from HIV-infected high viremic individuals, restored their ability to respond through their BCRs to both proliferate and differentiate into antibody secreting cells. Taken together these findings are important in defining potential targets for reversing the deleterious effects of FcRL4 on immune responses during chronic pathogen infections. We also characterized the effect of FcgammaRIIB engagement on the activation of human naive and MBCs. Using high-resolution imaging we showed that human MBCs are more robust than naive B cells at each step in the initiation of BCR signaling, including interrogation of antigen containing membranes, formation of sub-microscopic BCR oligomers and recruitment and activation of signaling-associated kinases. Despite their robust response to antigen, MBCs remain highly sensitive to FcgammaRIIB-mediated inhibition. These observations are important for understanding the regulation of memory antibody responses. Over the last year we initiated studies to use super-high resolution imaging to describe, at the 10-50 nm level, the spatial relationship between the BCR, CD19 and FcgammaRIIB in resting B cells and in B cells in which the BCR is ligated alone by Ag or Fcgamma is ligated by immune complexes. Our high resolution images showed that BCR ligation leads to the accumulation of BCRs and CD19 into the immune synapse and the initiation of signaling. Ligation of the FcgammaRIIB results in its accumulation in the immune synapse and the exclusion of the BCR and CD19. Thus, even at this level of resolution the spatial organization of the B cell receptors appear to be regulated by mechanisms that do not require direct coligation.